Clostridium perfringens (C. perfringens) is an anaerobic bacterium that is naturally found in soil, decaying organic matter, and as part of the normal gut flora of animals, including humans. C. perfringens is also the etiological agent for numerous clostridial diseases found in economically valuable domestic animals. C. perfringens produces a number of toxins that cause pathogenic effects in animals, including the alpha toxin, the beta toxin, the beta 2 toxin, the epsilon toxin, the theta toxin, the mu toxin, the delta toxin, the iota toxin, the kappa toxin, and the lambda toxin. Moreover, C. perfringens encodes other biologically active substances that can cause pathological effects, including: hyaluronidase, acid phosphatase, protease, collagenase, sulfatase and neuraminidase.
Different strains of C. perfringens are designated as biotypes A through E, depending on the spectrum of toxins that the particular bacterium produces [Justin et al., Biochemistry 41:6253-6262 (2002); McDonel, PHARMACOLOGY OF BACTERIAL TOXINS; F Dorner and J Drews (eds.) Pergamon Press, Oxford (1986)]. Initially, such typing was based on serologic neutralization assays in mice or guinea pigs. More recently, molecular typing methods employ polymerase chain reaction (PCR) targeted to genetic sequences that encode one of the numerous toxins of C. perfringens. 
Intestinal clostridiosis in horses has been correlated with high concentrations of C. perfringens Type A in the gut. Afflicted equine have a profuse watery diarrhea and a high mortality rate. C. perfringens Type A also has been linked to enteric disease in suckling and feeder pigs, with the symptoms including mild necrotizing enteritis and villous atrophy [Songer, Clin. Micro. Rev, 9(2):216-234 (1996)].
Clostridial diseases caused by C. perfringens are characterized by sudden death in well-fleshed birds with confluent fibrinonecrotic lesions (“Turkish towel”) in the small intestine (enteric forms), and/or C. perfringens-associated hepatitis with cholangiohepatitis, or fibrinoid necrosis in the liver. Afflicted birds undergo a rapid course of depression, diarrhea, and dehydration. Mortality ranges from 2% to 50%. Liver pathology leads to carcass condemnations at slaughter.
Necrotic enteritis (NE) is an example of a clostridial enteric disease caused by C. perfringens that leads to significant economic consequences in poultry. The disease is especially common in floor-reared broiler chickens from 2 to 10 weeks of age, though the disease also has been reported in turkeys and caged laying hens. Necrotic enteritis generally occurs in poultry either as a secondary disease, or in a situation in which the normal intestinal microflora are altered so as to allow the abnormal proliferation of pathogenic C. perfringens. The prevalence of subclinical necrotic enteritis is unknown, since the lesions can only be observed through post-mortem examination. However, reports of impaired feed conversion and reduced body weights have been attributed to the subclinical disease [Lovland and Kadhusdal, Avian Path. 30:73-81 (2001)]. Predisposing factors that lead to necrotic enteritis in both natural outbreaks and experimental models include: (a) coccidiosis, (b) migration of parasitic larvae, (c) feeds high in fish meal or wheat, and (d) immunosuppressive diseases. In addition, necrotic enteritis can be experimentally reproduced by: (i) providing animals feed contaminated by C. perfringens, (ii) administering animals vegetative cultures orally or into the crop, or (iii) intraduodenal administering of broth cultures of bacteria-free crude toxins to the animals.
C. perfringens Types A or C are the two biotypes that cause necrotic enteritis in poultry, with alpha toxin being the most common toxin detected [Wages and Opengart, Necrotic Enteritis. pp: 781-785, In: Disease of Poultry, 11th ed., (eds., Saif et al.), Iowa State Press, Ames, Iowa (2003)]. Indeed, greater than 90% of the C. perfringens isolates obtained from forty-two Type A infected fowl produced a lethal alpha toxin, along with sialidase, and the theta and mu toxins [Daube et al., AJVR 54:496-501 (1996)]. In addition, enterotoxin produced by C. perfringens Type A has been identified in chickens with necrotic enteritis and may play a role in the intestinal disease [Niilo, Can. J. Comp. Med. 42:357-363 (1978)].
Recently, it has been reported that C. perfringens Type A or Type C can encode the cpb2 gene [Gilbert et al., Gene 203:56-73 (1997)] and express its product, the beta 2 toxin. A strong correlation between the expression of beta 2 toxin and neonatal enteritis in swine has been observed [Bueschel et al., Vet Micro 94:121-129 (2003)]. The beta 2 toxin also has been implicated as a pathogenic factor in enteritis of horses and cattle. Bueschel et al., supra, further reported that 37.2% of the three thousand and twenty C. perfringens isolates that they obtained encoded the beta 2 toxin, and of the C. perfringens Type A isolates examined, 35.1% encoded the beta 2 toxin. The limited sample (n=5) of avian C. perfringens Type A field isolates were found to be ≈35% positive for the cpb2 genotype, and 40% of these also expressed the beta 2 toxin. In addition, employing PCR, Engstrom et al., [Vet Micro 94:225-235 (2003)] found that 12% of the C. perfringens isolates from chickens with hepatitis also were positive for cpb2. The enterotoxin of C. perfringens Type A has also been shown to be pathogenic in chickens, causing accumulation of fluid in a ligated intestinal loop model [Niilo, Appl. Micro. 28:889-891 (1974)].
Current efforts to control C. perfringens rely upon sanitary measures and placing antibiotics in the animal feed. The clostridial component of the disease responds well to antibiotics and is generally suppressed by antibiotic feed additives and ionophorous, anticoccidial drugs. However, antibiotics are costly and subject to increasing concerns related to the promotion of bacterial resistance.
Vaccination also has become an important control measure in domestic animals, since the course of many C. perfringens-associated diseases is rapid and often fatal. For example, U.S. Pat. No. 4,292,307 defines one “universal” multivalent vaccine prepared from toxoids of C. perfringens Type A, Type B, and Type D that further includes toxoids from Cl. oedematiens, and Cl. septicum toxoid. In addition, vaccines are commercially available that are often multivalent and consist of inactivated cells, toxins, or combinations of these two [see, Songer, Clin. Micro. Rev, 9(2):216-234 (1996)].
Vaccination of female livestock may also elicit passive protection of their subsequently born offspring. Passive protection of mammalian neonates against pathologic Clostridial infections relies upon the transfer of specific antibody in the form of colostral antibodies. For example, Smith and Matsuoka [Am. J. Vet. Res. 20:91-93 (1959)] employed an inactivated vaccine to inoculate pregnant sheep and reported maternally induced protection of young lambs against the epsilon toxin of C. perfringens. Passive immunity in young mammals typically lasts 2 to 3 weeks [Songer, Clin. Micro. Rev, 9(2):216-234 (1996)].
In contrast to suckling mammals, passive immunity in avians has several obvious shortcomings, most notably, the complete lack of the maternal antibodies obtained from milk. Though passive immunity is just one possible explanation for their observed correlation, Heier et al., [Avian Diseases 45:724-732 (2001)] did report that the survival of chicks was significantly higher in Norwegian Broiler flocks having higher titers of specific, naturally occurring maternal antibodies against C. perfringens alpha toxin than those flocks having low titers. In addition, Lovland et al. [Avian Pathology 33(1):83-92 (2004)] reported results that were consistent with modest passive protection of the progeny of hens that had been inoculated with vaccines based on C. perfringens Type A or Type C toxoids, as compared to the progeny of unvaccinated hens.
Heretofore, commercially significant protection from C. perfringens did not appear be attainable unless maternal antibodies for most, if not all of the pathologic components produced by C. perfringens bacteria were present in the inoculum. For example, vaccination of the dam with a product that does not contain enterotoxin only offers partial protection to piglets, and anti-epsilon toxin antibody in mother goats protects against death from toxemia, but not against enterocolitis [Songer, Clin. Micro. Rev, 9(2):216-234 (1996)].
Indeed, despite the tabulation of an impressive quantity of data regarding the various biotypes of C. perfringens and their corresponding toxins and deleterious bioactive substances, clostridial diseases in food producing animals remain a significant economic problem for farmers. Therefore, there is a need to provide additional means for protecting livestock against the pathological effects of C. perfringens. More particularly, there remains a need to provide a safe and effective vaccine against C. perfringens in poultry. Moreover, there is a need to provide a simple vaccine against C. perfringens that can be administered to female animals, especially fertile and/or pregnant female livestock, that will passively protect their offspring.
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